Assessment of cortical inhibition depends on inter individual differences in the excitatory neural populations activated by transcranial magnetic stimulation

Transcranial magnetic stimulation (TMS) is used to probe inhibitory intracortical neurotransmission and has been used to infer the neurobiological dysfunction that may underly several neurological disorders. One technique, short-interval intracortical inhibition (SICI), indexes gamma-aminobutyric acid (GABA) mediated inhibitory activity and is a promising biomarker. However emerging evidence suggests SICI does not exclusively represent GABAergic activity because it may be influenced by inter-individual differences in the specific excitatory neural populations activated by TMS. Here we used the latency of TMS motor evoked potentials (MEPs) to index these inter-individual differences, and found that a significant proportion of the observed variability in SICI magnitude was accounted for by MEP latency, r = − 0.57, r2 = 0.33, p = .014. We conclude that SICI is influenced by inter-individual differences in the excitatory neural populations activated by TMS, reducing the precision of this GABAergic probe. Interpreting SICI measures in the context of MEP latency may facilitate a more precise assessment of GABAergic intracortical inhibition. The reduced cortical inhibition observed in some neuropathologies could be influenced by reduced activity in specific excitatory neural populations. Including MEP latency assessment in research investigating SICI in clinical groups could assist in differentiating the cortical circuits impacted by neurological disorders.

were recruited from an Australian university and surrounding area via online and poster advertisements. Standard TMS exclusion criteria and pre/post safety screening procedures were applied 24,25 , including exclusion of potential participants with any self-reported history of neurological or psychological disorder, or current medical or recreational use of psychoactive drugs. The study was approved by the Deakin University Human Research Ethics Committee and all participants provided written informed consent in accordance with the Declaration of Helsinki.
Experimental protocol. Participants were seated in an adjustable chair with their right arm resting on a table positioned just above their lap. See Fig. 1 for the experimental procedure (described in detail below). Briefly, the experiment began with locating the stimulation site, then the relevant coil orientations were used to www.nature.com/scientificreports/ determine motor thresholds, apply single pulse TMS to assess MEP latency, and administer paired pulse stimulation to assess intracortical inhibition. Experiments were well tolerated by participants and no significant adverse effects were reported.
Transcranial magnetic stimulation. Monophasic TMS pulses of 100 µs rise time and 1 ms duration were generated with a Magstim Bistim 2 system (Magstim United Kingdom) and delivered with a 70 mm Magstim figure-8 coil via the Bistim connecting module. Hotspot and thresholds were assessed via single pulses delivered in Bistim mode, latency and SICI blocks were assessed in independent Bistim triggering mode 27 . The left M1 FDI hot spot for the contralateral right hand was located in accordance with previous guidelines 26  AMT. LM latencies were assessed with 10 trials in active muscle, and a stimulus intensity of 150% AMT in order to increase the likelihood of evoking an MEP commencing with a D wave 30 . Here, as previously 16 , 10 trials at this higher stimulus intensity provided sufficiently reliable latency estimates 31 . MEP onset was also assessed in the unconditioned test pulses used to calculate SICI. These comprised 20 trials delivered at SI1mV in resting muscle, using both PA and AP current.
SICI assessment. SICI was assessed in resting muscle in the PA and AP current directions. The CS was delivered at 90% AMT, followed 2.5 ms later by the TS delivered at SI1mV 32,33 . The intensities were determined with reference to the relevant thresholds established in each direction. Twenty SICI conditioned trials and 20 single pulse SI1mV trials were interleaved and jittered with 5, 6, and 7 s inter-stimulus intervals in pseudorandomized order.
Data analysis. Because voluntary contraction can substantially alter the composition of the CSV 34 , and also alter SICI 35 , any trials with EMG amplitude greater than 20 µV in the 100 ms prior to the stimulus (24%, 545 of 2240 trials) were excluded from SICI calculations. Because SICI calculation relies on averaged amplitudes, we made a pragmatic decision that any average amplitudes calculated from less than 5 MEPs were likely unreliable 36 and therefore were not calculated. This meant that in any block of 20 SICI trials (conditioned or unconditioned) the median number of trials averaged was 18 (min 5, max20). We ultimately calculated SICI PA magnitudes for 23 participants and SICI AP magnitudes for 25 participants. MEP latencies were calculated for each participant and each group of latency trials using a custom Matlab script. EMG signals for the block were averaged 8 and the latency was identified as the first timepoint following 15 ms post stimulus where the averaged signal exceeded the mean plus 5 standard deviations of the averaged signal in the 100 ms prior to the stimulus. Any pulses that did not reach their individual 5 standard deviation threshold (14%, 350 of 2520 trials) were excluded from the averaging. Because latency can be altered by voluntary contraction 23 any resting latency trials with EMG amplitude greater than 20 µV in the 100 ms prior to the stimulus (24%, 545 of 1120 trials) were also excluded from the averaging of EMG signals for resting PA and AP latency detection. Because MEP latency is more reliable than MEP amplitude 37 we made a pragmatic decision that latency should only be calculated where at least 3 trials were available to create the averaged EMG signal. This meant that in any block of 20 latency trials the median number of trials averaged was 16 (min 3, max 20), and for LM latency all blocks were averaged from 10 trials. We ultimately obtained PA and LM active latencies for all (28) participants, AP active and AP resting for 27 participants, and PA resting for 26 participants. The averaged traces and detected latencies were plotted for visual inspection revealing 7 clear failures (of the 136 plots), due to noise or dc drift in the signal. These latencies were corrected by manually adjusting the onset to the first subsequent point clearly associated with the MEP response where the EMG signal exceeded the mean plus 5 SD of the pre-pulse EMG 7,16 . The mean plus 5 SD threshold was chosen to keep the failed detections, and hence the manual adjustments, to a minimum in order to maximize the objectivity of the extracted latency metric. We also made a further check on our automatically detected laten- www.nature.com/scientificreports/ cies (detailed and reported in the "Supplementary material") and confirmed that they were similar to the onsets observed via visual inspection of overlay plots of all trials for each block. For each participant, active LM latency (the indicator of D wave latency) was subtracted from both resting and active PA and AP latencies to create latency difference metrics 16 . This provided an indication of whether the CSVs evoked in each direction (PA and AP) and condition (resting or active) tended to commence with earlier (i.e. smaller values, closer to D wave latency) or later (i.e., larger values, further from D wave latency) I waves. The resulting latency difference metrics were PA-LM ACT , AP-LM ACT , PA-LM REST , and AP-LM REST .
For each participant and current direction, (excluding trials containing EMG activity as explained above) SICI was calculated by expressing the average of the conditioned MEP amplitudes as a percentage of the average unconditioned amplitudes (i.e. (conditioned amplitude/unconditioned amplitude) × 100, 100 = no inhibition, below 100 = inhibition). The resulting metrics were SICI PA and SICI AP .
Statistical analysis. Assumption testing, analyses, post-hoc testing, and adjustments for multiple comparison are detailed here in order to simplify the presentation of the results section that follows. Firstly, three separate preliminary comparisons were conducted to confirm our measurements were in accordance with values previously reported 16,23 . These comparisons were of the motor thresholds (AMT PA , AMT AP , AMT LM , SI1mV PA , and SI1mV AP ), raw latencies (PA ACTIVE , AP ACTIVE , LM ACTIVE , PA RESTING , and AP RESTING ), and latency difference scores (PA-LM ACT , AP-LM ACT , PA-LM REST , and AP-LM REST ). Quantile-standardized residual plots suggested that motor thresholds, raw latencies, and latency difference scores were approximately normally distributed. However none in this series met the assumption of sphericity assessed via Mauchley's test therefore repeatedmeasures ANOVA with Greenhouse-Geisser correction applied was used for comparisons. Post-hoc pair-wise comparisons with Tukey's adjustment were then conducted (see Supplementary Material Tables S1-S3). SICI PA and SICI AP difference scores were normally distributed, as assessed via a Shapiro-Wilk test and density plot, therefore a paired-samples T-test was used to compare them. For these preliminary analyses effect sizes are reported using Hedge's g av to account for the inherent correlation between these intra-individual effects, and to facilitate any future use of these results 38 .
Scatterplots were assessed prior to the main correlational analyses and indicated no outliers or non-linear relationships were present. Linear correlations (Pearson's) were used to test for a relationship between SICI PA , SICI AP and each of the four latency difference metrics. We note some positive skew (a possible floor effect) was present in SICI AP . This may suggest a degree of caution in the interpretation of the SICI AP Pearson's coefficients. Familywise error was controlled separately for the SICI PA and SICI AP comparisons with Holm-Bonferroni adjusted p values. Statistical analyses were performed using RStudio Version 1.4.1106.

Results
Descriptive statistics for SICI magnitudes, MEP latencies, and motor thresholds are reported in Table 1.
Cortico-spinal excitability-the effect of current direction and stimulus intensity. Repeatedmeasures ANOVA indicated that there were significant differences in motor thresholds (AMT PA , AMT AP , AMT LM , SI1mV PA , and SI1mV AP ), F(2.48, 67.05) = 148.82, p < 0.001, ω 2 = 0.84. Post-hoc comparisons with Tukey's adjustment indicated there were significant differences between all thresholds (see table S1 in "Supplementary Material"). The lowest was AMT PA followed in increasing order by AMT LM, AMT AP , SI1mV PA , and SI1mV AP .  Table S2) indicated raw latencies were significantly different across all current directions, being shortest with LM current and longest with AP current. There was no significant difference between resting and active PA, or between resting and active AP latencies, however we note these were obtained under different conditions (see "Supplementary Material" for discussion).

Short-interval intracortical inhibition
There were significant differences in latency difference metrics (

Discussion
The current study sought to further characterize the nature of the relationship between SICI and inter-individual variations in the neural populations activated by TMS (as assessed using MEP latency difference metrics). The relationship was examined, using both PA and AP current directions, by assessing MEP latency in active and resting muscle, and assessing SICI magnitude in resting muscle. Here we used conventional SICI where, because the test stimulus intensity is held constant, the composition of the test CSV is likely less variable than in the ttSICI examined previously. Our preliminary analyses indicated that mean PA-LM ACT and AP-LM ACT latency differences (1.7 ms and 3.4 ms respectively, see "Supplementary Material" for further discussion) were consistent with values previously used to index differences in I wave recruitment 13,16 . In line with our expectations, our  www.nature.com/scientificreports/ results show that greater SICI PA is associated with longer AP-LM MEP latency difference, explaining up to 33% of the inter-individual variability in conventional SICI. The relationship between SICI and latency difference seen here, and in previous work, has significant implications (detailed below) for the interpretation of each measure. Contrary to our expectations, SICI AP was not associated with MEP latency difference. We found that 33% of the observed individual variability in intracortical inhibition assessed with PA current was explained by AP-LM latency difference. As expected, longer AP-LM latencies were associated with greater SICI PA , supporting the idea that individual levels of SICI depend on individual tendency toward earlier or later I wave recruitment. Both resting and active AP-LM latencies were closely associated with SICI PA . To our knowledge, this is the first report of an association between AP-LM REST latency and SICI PA , suggesting that, for our current purpose, probing MEP latency at SI1mV in resting muscle may be equally as useful as the more  www.nature.com/scientificreports/ common 110% AMT probe used in active muscle. Our findings also support the previous account of a correlation between AP-LM ACT latency and ttSICI PA 8 , and demonstrate here that the correlation is also apparent when the TS is held constant. While AP-LM latencies do not directly reflect the PA TS used in SICI PA , they are used to infer the extent of an individual's later I wave recruitment. It seems logical that SICI's selective inhibition of I3 and later waves, combined with SICI's lack of impact on early I waves, could be driving the relationships we observed. We did not detect statistically significant relationships between SICI PA and PA-LM latencies. Perhaps PA-LM latency is not sufficiently sensitive to the late I waves inhibited by SICI, however we note the scatterplots and coefficients appear to be in agreement with the direction of the relationship we found for AP-LM latencies.
Unlike with SICI PA , we observed no linear relationships between SICI AP and any latency difference measure. This was in contrast to a previous report of an association between active AP-LM latency and ttSICI AP 7 . We acknowledge that our study may be limited by the possibility of a floor effect being present in our conventional SICI AP , as evidenced in the scatterplots and Fig. 4, which may have obscured any relationship between SICI AP and latency difference. Alternatively, the absence of an association between SICI AP and latency difference measures could be because assessing SICI with AP current avoids early I wave recruitment that would otherwise mask assessed inhibition.
Our findings of an association between conventional SICI PA and AP-LM latencies represent the third report of a significant, likely neurophysiologically-driven, and arguably underappreciated relationship between SICI and MEP latency difference. Here we demonstrate for the first time that this association is present for conventional SICI, where test stimulus CSV composition is held relatively constant. Accounting for this relationship could contribute to a more individualized understanding of both GABAergic inhibitory activity and TMS preferential recruitment of distinct neural populations. The major implication here is that interpreting SICI in the context of latency difference could increase the accuracy and utility of the measure. For example, in individuals with short latency difference, who therefore have early I waves present in the CSV, the absence of SICI, i.e. no inhibition of the SICI test pulse, could mean that no later I waves were present to be inhibited, or alternatively that GABAergic activity was not apparent. In these individuals, a test pulse that reveals inhibition indicates the presence of later I waves, but the assessed SICI may only provide a diluted measure of GABAergic activity due to the presence of unaffected early I waves. However, in individuals with longer latency difference who therefore do not have early I waves present in their CSV, the extent of inhibition of the test pulse may reflect a more accurate index of GABAergic activity.
Greater precision in SICI assessment may be important in clinical investigations that report reduced SICI in a range of neurological disorders [2][3][4]39 . It is possible that the use of long latency difference subgroups may allow for more accurate comparisons of SICI function in clinical and healthy subjects. We also note that differences in www.nature.com/scientificreports/ I wave recruitment could be contributing to the SICI dysfunction identified in clinical populations. The inclusion of latency difference assessment in future SICI research could speak to this contribution, potentially increasing our understanding of the mechanisms underlying the reduced SICI observed in some clinical groups. A further implication of our findings is that conventional SICI PA may provide a diluted index of GABAergic inhibitory activity. As suggested previously 40 , SICI AP may deliver a more accurate assessment of intracortical inhibition by avoiding the unaffected early I wave recruitment that might mask assessed inhibition. There are also implications for interpreting MEP latency. A growing body of research suggests that later I wave recruitment (assessed via MEP latency difference) is associated with TMS-induced neuromodulation outcomes, and with learning 16,41-44 , but because latency can only reflect the first component of the CSV, short latencies cannot speak to the presence of later I waves. In individuals with short latency difference the presence of SICI could indicate that later I waves are also present in their CSVs. We suggest that including SICI assessment in future research could, at the individual level, facilitate a more detailed understanding of how preferential TMS recruitment of distinct neural populations impacts TMS neuromodulation outcomes.
Our study used single and paired pulse TMS to examine the relationship between MEP latency difference and SICI assessed using PA and AP current. Latency difference was used to indicate whether individual motor response to TMS tended to commence with earlier or later I waves. We found that a significant proportion of the observed variability in PA SICI magnitude can be accounted for by MEP latency difference, reflecting individual differences in the neural populations preferentially activated by TMS. However, MEP latency difference did not account for the variability we observed in AP SICI. We suggest that interpreting SICI measures in the context of individual I wave recruitment patterns will contribute to more precise assessment of GABAergic intracortical inhibition, that AP SICI could more accurately reflect inhibitory processes, and that accounting for SICI could enhance our understanding of the relationship between MEP latency difference and TMS neuromodulation outcomes.

Data availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.